Role of TIMPs in Hematopoietic Stem Cell Biology

描述（由申请人提供）：造血干细胞（hsc）与骨髓（BM）细胞外基质（ECM）密切相关，是干细胞“生态位”的一部分。与ECM的相互作用可以调节造血干细胞的增殖/分化，移植能力对于移植后正常的再繁殖活动至关重要。此外，在发育过程中，原始造血干细胞必须适当地从主动脉-性腺-中肾（AGM）和卵黄囊区域迁移，在胎儿肝脏中成熟和扩展为最终的造血干细胞，并最终迁移到新生儿的骨髓中。基质细胞分泌的生长因子可以调节造血干细胞在发育和成年期间与ECM的相互作用。因此，如果造血干细胞要用于临床，了解细胞因子功能的分子调节因子是很重要的。基质金属蛋白酶（MMP）家族的成员在生长因子刺激后转录上调。MMPs是锌依赖性蛋白酶，可切割多种ECM成分。具体来说，在细胞因子刺激后，巨噬细胞、淋巴细胞和CD34 +细胞中的MMP-9上调。中性粒细胞释放MMP-9也与HSC动员高度相关。我们提议测试基质金属蛋白酶的天然抑制剂，即基质金属蛋白酶的组织抑制剂（TIMPs）是否通过作为基质金属蛋白酶活性的负调节因子来维持基质金属蛋白酶生态位中的蛋白水解平衡。除了抑制MMP活性外，TIMP-1还具有不依赖于MMP的促红细胞生长活性，可以刺激生发中心B淋巴细胞的发育。我们已经生成了表达人TIMP-1的逆转录病毒载体，并在体外发现了一种新的通过自分泌机制介导的小鼠髓系M1细胞分化抑制活性。我们的目的是确定：1)体外M1细胞中人TIMP-1活性的结构/功能关系2)人TIMP-1在小鼠骨髓造血干细胞中的组成性表达是否会改变长期的再填充活性和归巢/植入特性3)正常造血是否需要内源性TIMP-1在小鼠造血干细胞或基质细胞中表达。我们提出TIMP-1具有mmp依赖和mmp独立的功能，通过限制干细胞的募集和动员来调节造血，以应对造血应激。干细胞为基础的治疗具有巨大的前景，这些研究可以通过直接影响HSC的存活和增殖，为增加扩增或胚胎来源的HSC的植入，限制髓系炎症细胞组织的浸润，或抑制肿瘤生长和血管生成提供新方法。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) closely associate with the bone marrow (BM) extracellular matrix (ECM) as part of the stem cell "niche". Interaction with the ECM can regulate the proliferation/differentiation of HSCs and the engraftment ability is critical for normal repopulating activity following transplant. Also, during development, primitive HSCs must migrate appropriately from the sites of the aorta-gonad-mesonephros (AGM) and yolk sac regions, to mature and expand as definitive HSCs in the fetal liver, and finally move to the BM in the newborn. Growth factors secreted from stromal cells can regulate the interaction of HSCs with the ECM during development and in the adult. Therefore, it is important to understand the molecular regulators of cytokine function if HSCs are to be manipulated for clinical use. Members of the matrix metalloproteinase (MMP) family are transcriptionally upregulated following growth factor stimulation. MMPs are zinc dependent proteases that cleave a wide range of ECM components. Specifically, MMP-9 is upregulated in macrophages, lymphocytes, and CD34 + cells following cytokine stimulation. MMP-9 release from neutrophils is also highly associated with HSC mobilization. We propose to test whether natural inhibitors of the MMPs called tissue inhibitors of matrix metalloproteinases (TIMPs) maintain the proteolytic balance in the BM niche by acting as negative regulators of MMP activity. In addition to the MMP inhibiting activity, TIMP-1 has MMP-independent erythroid growth promoting activity and can stimulate development of germinal center B lymphocytes. We have generated retroviral vectors expressing human TIMP-1 and found a novel differentiation inhibiting activity on murine myeloid M1 cells in vitro that was mediated through an autocrine mechanism. We aim to determine: 1) The structure/function relationship for human TIMP-1 activity in M1 cells in vitro 2) Whether constitutive expression of human TIMP-1 in murine BM HSCs alters long-term repopulating activity and homing/engraftment properties 3) Whether endogenous Timp-1 expression is required in murine HSCs or stromal cells for normal hematopoiesis. We propose that TIMP-1 has MMP-dependent and MMP-independent functions that regulate hematopoiesis by limiting the recruitment and mobilization of stem cells in response to hematopoietic stress. Stem cell-based therapies hold tremendous promise and these studies could lead to novel approaches for increasing engraftment of expanded or embryonic-derived HSCs, for limiting infiltration of tissues with myeloid inflammatory cells, or for inhibiting tumor growth and angiogenesis, through direct effects on HSC survival and proliferation.